Clonal Spread of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 in Chinese Pediatric Patients

ABSTRACT Klebsiella pneumoniae often causes life-threatening infections in patients globally. Despite its notability, little is known about potential nosocomial outbreak and spread of K. pneumoniae among pediatric patients in low- and middle-income countries. Ninety-eight K. pneumoniae strains isolated from pediatric patients in a large general hospital in China between February 2018 and May 2019 were subjected to nanopore and Illumina sequencing and genomic analysis to elucidate transmission and genetic diversity. The temporal distribution patterns of K. pneumoniae revealed a cluster of sequence type 11 (ST11) strains comprising two clades. Most inferred transmissions were of clade 1, which could be traced to a common ancestor dating to mid-2017. An infant in the coronary care unit played a central role, potentially seeding transmission clusters in other wards. Major genomic changes during the outbreak included chromosomal mutations associated with virulence and gains and losses of plasmids encoding resistance. In summary, we report a nosocomial outbreak among pediatric patients caused by clonal dissemination of KPC-2-producing ST11 K. pneumoniae. Our findings highlight the value of whole-genome sequencing during outbreak investigations and illustrate that transmission chains can be identified during hospital stays. IMPORTANCE We report a nosocomial outbreak among pediatric patients caused by clonal dissemination of blaKPC-2-carrying ST11 K. pneumoniae. Strains of various sequence types coexist in the complex hospital environment; the quick emergence and spread of ST11 strains were mainly due to the plasmid-mediated acquisition of resistance genes. The spread of hospital infection was highly associated with several specific wards, suggesting the importance of genomic surveillance on wards at high risk of infection.

Liu and colleagues describe the clonal spread of carbapenem-resistant Klebsiella pneumoniae ST11 in Chinese pediatric patients throught an extended genomic analysis including phylogeny and mobile element content (antibiotic resistance and virulence genes). Results, in particular the use of phylodynamics in outbreak context, are interesting and this paper is an example of the use of multiple genomic tools to understand outbreak dynamics, in particular for Klebsiella pneumoniae in children hospital context. Apart from these aspects, many formulations and statements are inadequate (see section "Technical and content comments"). Regarding the style, the manuscript would benefit from review by a native speaker.
Reviewer #3 (Comments for the Author): The study investigates the population structure and phylogeny of 98 Klebsiella pneumoniae isolates obtained from pediatric patients in a Chinese hospital between February 2018 and May 2019. The authors reported the clonal spread of KPC-2 producing ST11 K. pneumoniae strains that were clustered into 2 clades. The study described the plasmid content in the 2 clades complete with resistance genes. The authors used these findings to explain the higher transmissibility among clade 1 isolates relative to clade 2 isolates. The study calls for a wider use of genomic tools to identify outbreaks.
Minor comments were observed: The manuscript needs minor English revision and editing.
-Line 64: Would the authors please explain the importance of carbapenem as last resort agent in the treatment and hence the global concern as a result of carbapenem resistance development? -Line 117: was the DNA library prepared at the sequencing company? Please give some details about Illumina library prep.
-Line 124-125: Would the authors please give the details of quality check and trimming of the reads? -Would the authors give more details about Fig. 3c as it is not clear how they inferred the substitutions per site per year from the figure.
-Lines 297-297: Fosfomycin and macrolide susceptibility data are not shown in the table.
-Line 300: Do the authors mean a statistically significant difference? -Lines 304-306: The authors would better comment on the almost complete absence of these genes in the ST11 strains.
-Line 306: The authors would better specify that they mean IncFII.pHN7A8._1 -Line 306-308: "All ST11 strains carried the ColRNAI and IncFII plasmid replicons, which were highly correlated with the presence of blaKPC-2, rmtB, and blaTEM-1B genes" the isolates also carried blaSHV.11_1, blaSHV.155_1 as well as fosA_3 coding for Fosfomycin resistance and oqxA_1 and oqxB_1 coding for fluoroquinolone resistance. -Line 308: Looks like only 2 clade 1 strains (and not 3) co-carried blaKPC-2 and mcr-9. -Line 310: Looks like the three mcr-9 positive strains carried the pKPC.CAV1321_1 and not repB_KLEB_VIR plasmid replicon. Lines 311-313: the same can be said about ST76 strains and IncX3_1 plasmid replicon and blaNDM.1_1, blaSHV.12_1, fosA_3, oqxA_1 and oqxB_1. Would the authors please comment on Fig 5B at the end of the results? From Fig 3D and Fig 5B, it seems that strain xz163 evolved from a distant parent xz061 and acquired a 4th plasmid then further evolved to xz168 and xz181. Lines 314-316: Please mention Additional file 8 Figure S6 here. Lines 371-372: Figures 3D and 3E show the transmission of clade 1 but not clade 2, so unless the authors are referring to other figures here, would they just mention that these results are not shown?
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Line 328: Add "probably" first of "indicate that…." Line 347: remove "the" at the beginning of the line Line 356-359: this phrase is not clear. Rewrite.
Line 364: the term "obtained" is not appropriate Line 381-383: this concept is trivial. Rewrite or remove it Line 389: replace with "plasmids in hospital strains"    Response 24: 265 Thanks very much for the reviewer's comments. 266 We have changed "In addition, we identified 5 genes with more than 267 one SNP." with "In this case, a recent population expansion was 268 more likely to happen. All the mutations were annotated according to 269 their genome positions, from which 5 genes were identified with 270 more than one mutation, so we further analyzed the mutation type   Figure S5). However, only three genes 531 (blaKPC-2, rmtB, and blaTEM-1B) among them were found located 532 on the plasmid contigs indicating that they might be located the two were not shown. 602 We have revised the description in the resubmitted manuscript.  Your paper has been further revised and all the reviewers suggest other modifications and to address completely previous comments Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. The bacterial isolation and AST have been simplified rather than elaborated and explained further. How the bacteria were isolated, at what point identification and antimicrobial susceptibilities were performed are not explained. A review of the electronic medical records should not be explained in the methodology for bacterial isolation and antimicrobial susceptibility. Access to the patients' personal medical files should be explained in the ethical approval as well.
The results of the hand hygiene audit were not provided, instead a detailed explanation of the infection prevention measures was provided. Hand hygiene audit results would indicate the compliance of staff in performing hand hygiene in the wards and the audits are usually conducted regularly by the infection prevention staff.
Reviewer #2 (Comments for the Author): Authors improved the quality of the manuscript, adding the experimental and methodological information required by reviewers. Despite this, some formulations and statements remain still inadequate or need to be synthetized. Regarding the style, the final manuscript, in particular some new statements, would benefit from review by a native speaker. A control of the correct use of gene nomenclature (italic, subscript character ect.) is needed.
Reviewer #3 (Comments for the Author): Would the authors kindly add their response to point 37 to the manuscript? In the authors' response to point 42, they explained that they carried out Pearson's Chi-squared Test for the strains carrying the blaKPC-2 gene between ST11 and non-ST11 isolates. The authors are kindly requested to add the statistical analysis to the methods section?
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Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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REVIEWER n2
1 Authors improved the quality of the manuscript, adding 2 experimental and methodological information required by reviewers.  whole genome" (line 147) and "GTR+ was selected as the best evolutionary model by using Modeltest-ng v0.1.7" (line 150-151).

Response 10:
Thanks very much for the reviewer's comments, we have revised the two sentences as suggested.

Point 11:
The reference you mentioned (42)  In our analysis (Additional file 5 Figure S3), the strains were divided into two groups, one group had less than 30 SNPs difference among each other, while the other group had more than 43 SNPs. Thus, we had reduced threshold and set the value to 30.
We had added the reference as suggested and revised the description as "Pairs of genomes within a distance less than 30 SNPs were considered as the same transmission cluster (Fig. 3B, Additional file 5: Figure S3A)" (Lines 294-296)

Point 12:
Lines 171-173: in order to simplify the text, cancel "The slope of the regression was positive, and the p-value is 0.0006, showing that the genomic data reflect strong temporal signal.

Response 12:
Done as suggested.

Point 13:
Lines 322-328: in order to simplify the text, substitute only with "Most of the genes showed negative Tajima's D values (Additional file 6: Figure S4), in particular 5 genes with more than one SNP, suggesting a possible negative selection or a recent population expansion."

Response 13:
Done as suggested.
"Most of the genes showed negative Tajima's D values (Additional file 6: Figure S4), in particular 5 genes with more than one SNP, suggesting a possible negative selection or a recent population expansion." (Lines 333-336)

Point 14:
Lines 359-375: in order to simplify the text, substitute with "However, only three genes (bla KPC-2 , rmtB, and bla TEM-1B ) among them were found located on plasmid contigs. In addition, two Clade 1 strains co-carried bla KPC-2 gene and mcr-9 gene, which were located on IncFII pHN7A8.1 plasmid and IncHI2/IncHI2A plasmid respectively. IncHI2/ IncHI2A and pKPC.CAV1321_1 plasmid replicons were found only in the three mcr-9 positive strains. Seven of the eight ST76 strains carried the IncX3_1 plasmid replicon which appears to be associated with the presence of several resistance genes including bla NDM.1_1 , bla SHV.12_1 , fosA_3, oqxA_1 and oqxB_1 (Additional file 7 Figure S5). However, only bla NDM.1_1 gene was found located on plasmid contigs.

Point 16:
Lines 423-426: Replace with "Based on the spatio-temporal analysis, it was found that the ST11 was not the most abundant ST type at the beginning of the collection period, but it increased gradually becoming the most common ST type in collected strains.

Reviewer #3 (Comments for the Author):
Point 18: Would the authors kindly add their response to point 37 to the manuscript?

Response 18:
Thanks very much for the reviewer's comments, we have added the response to point 37 to the revised manuscript. In the authors' response to point 42, they explained that they carried out Pearson's Chi-squared Test for the strains carrying the blaKPC-2 gene between ST11 and non-ST11 isolates. The authors are kindly requested to add the statistical analysis to the methods section?

Response 19:
Thanks very much for the reviewer's comments, we have added the statistical analysis to the revised methods section.
"Pearson's Chi-squared Test was performed for the strains carried the bla KPC-2 gene between ST11 and non-ST11 multi-locus sequence types, the X-squared and p-value were calculated to infer statistically significant difference." (Lines 201-204) I am pleased to inform you that your paper has been accepted for publication Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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